Large-scale production of antibodies for biopharmaceutical applications involves the use of cell cultures that are known to produce antibodies, and antigen-binding portions thereof, exhibiting varying levels of heterogeneity. This heterogeneity may comprise isoforms of the antibody wherein the difference between the isoforms is dependent on charge. Some of these charge variants (or isoforms) may lead to decreased product efficacy and stability. Alternatively, adding a basic overall charge to an antibody may contribute positive attributes to the antibody such as increased tissue penetration.
One source of antibody heterogeneity involves C-terminal lysine residues, such as those typically found on the heavy chains of antibody molecules, which can be lost during both the purification process and/or storage of the final composition, resulting in compositions comprising antibody species that can vary at their C-terminus as to whether a lysine residue is present. For example, C-terminal lysines can potentially be present on both the heavy chains of an antibody (Lys 2), on either one of the heavy chains (Lys 1), or neither of them (Lys 0). Since lysine carries a positive charge, antibodies lacking the basic C-terminal lysine(s) differ in their charge state from ones that contain the lysine, so that the distribution of lysine variants (% Lys 0, % Lys 1, % Lys 2 of the total Lysine Sum) can be detected by ion-exchange chromatographic methods, for example, using a ProPac WCX-10 Weak Cation-Exchange column for the high-resolution separation of protein isoforms (Dionex, Calif.), and subsequently quantified.
The development of compositions comprising antibodies, or antigen-binding portions thereof, with either higher or lower levels of lysine variants to increase efficacy and stability of antibody products is an important, to date unmet, need in the biopharmaceutical industry.